P1315: ENGRAFTMENT AND MOBILIZATION OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS ALIGNS WITH DYNAMIC OSCILLATION OF TETRASPANIN CD9
Authors: W. H. Ng, C. Zhang, H. Wang, K. Y. Y. Chan, K. T. Leung
Journal: HemaSphere (2022)

Background: Entry and exit of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche require highly orchestrated molecular programs. Our group previously demonstrated the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis controlling homing of human CD34+ HSPCs but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remain largely unknown. Aims: This study was designed to (1) map the fate of HSPC engraftment following CD9 neutralization; (2) track the changes of CD9 expression during hematopoietic reconstitution and mobilization; (3) validate the oscillation of CD9 on donor HSPCs residing in the BM and circulation; and (4) address the mechanisms of CD9 governing HSPC trafficking. Methods: Cord blood (CB) CD34+ cells were exposed to a CD9 neutralizing antibody prior to infusion into irradiated NOD/SCID mice, with HSPC engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitor) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was determined by flow cytometry and compared with those before infusion or those undergone G-CSF-induced mobilization. The expression of CD9 were further characterized on HSPCs from normal donors in pairwise BM and peripheral blood (PB) samples, and on those in G-CSF-mobilized peripheral blood (MPB). Activities of the integrin VLA-4 (an established binding partner of CD9) were evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results: A short exposure of CB CD34+ cells to the CD9 neutralizing antibody substantially reduced their engraftment in the BM by 64.2% denoted by the human CD45+ population (P=0.003). No bias in lineage commitment was reflected upon CD9 blockade, suggesting engraftment of HSPCs is affected in general but not limited to specific subset of hematopoietic precursors. Comparing with those before transplantation, CD9 expression was significantly elevated on engrafted CD34+ cells retaining in the BM from 9.6% to 91.4% (P<0.001). Contrarily, G-CSF administration downregulated CD9 expression on BM CD34+ cells from 73.4% to 33.5% (P<0.001) where the decline was observed in both primitive stem cells and committed progenitors, indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, analysis of steady-state HSPCs from healthy donors showed that BM-resident CD34+ cells expressed a higher level of CD9 than those in the circulation (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was witnessed on BM CD34+ cells (47.7%; n=16) than those from MPB donors (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure (P=0.006), revealing a dynamic regulation. Subpopulation analysis of BM CD34+ cells dictated preferential binding of CD9+ cells to soluble VCAM-1 comparing with CD9- cells (51.4% vs. 25.9%, P<0.001; n=10). BM CD34+ cells also had a superior VCAM-1 binding capacity than the MPB counterpart (37.7% vs. 4.1%, P=0.014), suggesting CD9 may regulate HSPC trafficking via modulation of integrin activities. Summary/Conclusion: HSPC engraftment and mobilization coincide with dynamic oscillation of CD9 expression and integrin VLA-4 activities within the BM microenvironment. The current study has added impetus to our understanding on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.